Hi everybody,
I have (h)MeDip data from two different cell types I would like to compare to identify D(h)MR between the two cells. I used MACS 2 to call peaks and calculated a enrichment value according to this formula:
which is more or less the log2 fold ration of sequencing reads for a peak between antibody treated sample and non-antibody treated control (normalized by total number of reads). To compare now my two cells I calculated the log2fold change of the mean of the enrichment value (I have all experiments in triplicates) of the two cell types. Using this strategy I identify several regions with great GO terms of the nearest gene (which makes absolutely sense in the biological context of the experiment).
However I calculate a log2fold change with numbers already on a log2fold scale, sounds a bit weird for me (however iam a biologist and have no clue about mathematics in general ;) ). Furthermore i tried simply to calculate the fold change and setting up new cutoff parameters comparable to the previous used on the log2 scale, however i loss a lot of potential DMR regions.
Therefor can i continue to calculate the log2fold ration of the two enrichment values or is this simply wrong ? Alternatives ?
Thanks a lot,
Flo
Why don't you just not take the log2 of the fold change? (when you take a ratio b/w sample and control)
Sorry Iam still not sure which method I should use. Is the following pipeline correct if I want to identify differential methylated region between celltype A and celltype B (especially about 6 Iam not sure).
1. Call Peaks using MACS (for each cell type separately)
2. Merge the peak patterns
3. count the number of reads belonging to a certain peak for each cell type separately (AB-treated sample and Input)
4. Calculate the enrichment value using
for both cell types separately (more or less a log2 ration of seq. depth normalized reads in sample vs seq depth normalized reads in Input)
5. subset peaks which reach at least in one of the two cell types a certain enrichment value threshold (to get ride of peaks called by MACS which have only a low enrichment in both cell types)
6. for each of the filtered peaks I subtract the seq. depth normalized Input reads from the seq. depth normalized sample reads for each cell type separately
7. Using this values to calculate a log2 fold ration between celltype A and B
Is this correct (especially step 6) ?
Right now i do the same until step 5 but instead of going back to the raw reads I substract the enrichment value of cell B from cell A to obtain a value which expressed the differences of methylation of a genomic location (peak) between the two cell types.
Thanks for advises,
Flo